Aspergillus species, A. fumigatus, A. flavus and A. niger are the causative agents in human “Aspergillosis” and are also pathogens damaging Agricultural crops. They induce a variety of Aspergillus induced clinical conditions in immunocompetent and immunocompromised hosts. Depending on the host's immunity and the virulence of the clinical spectrum varies from aspergilloma, allergic Aspergillus sinusitis, allergic bronchopulmonary aspergillosis [ABPA], and hypersensitivity pneumonitis and invasive aspergillosis (Agarwal R. Allergic bronchopulmonary aspergillosis. Chest. 2009 March;135(3):805-26). These infections are often and progress fast and eventually fatal in immunocompromised patients. For example, pulmonary and cerebral aspergillosis has mortality rates of 86 and 99% respectively, even when adequately treated (Marr K A, Bowden R A. Fungal infections in patients undergoing blood and marrow transplantation. Transpl Infect Dis. 1999 December; 1(4):237-46). Allergic bronchopulmonary Aspergillosis in immunocompetent persons is often diagnosed by serological tests based on Aspegillus antigens and the specific IgE and IgG antibodies in the serum samples. Serodiagnostic tests based on Antigenic peptides and antigens are reported (Denning D W. Therapeutic outcome in invasive aspergillosis. Clin Infect Dis. 1996 September;23(3):608-15). In case of invasive Aspergillosis, particularly in immunocompromised host, the antibodies are present either negligible quantities or absent. Hence detection of circulating antigen or pathogen in clinical samples is the best strategy. This necessitates the development of dependable, specific methods for the detection of pathogens in clinical samples. In view of this efforts are made to develop more sensitive reagents and protocols for detection of important Aspergillus species in clinical samples.
Gene based methods are reported in the literature for detection of A. fumigatus, A. flavus and A. niger for clinical samples. They are mainly based on ITS regions of Aspergillus species which are genus specific (Abdin M Z, Ahmad M M, Javed S. Advances in molecular detection of Aspergillus: an update. Arch Microbiol. 2010 June;192(6):409-25. Epub 2010 Apr. 1). Further some of the important Aspergillus species such as Aspergillus fumigatus has been reported to develop resistance to Amphotericin and itraconazole. The need for a rapid test to identify Aspergilli to the species level, to assist in the selection of appropriate drugs for the treatment of clinical Aspergillus infections is also of high importance. Nonculture-based methods are increasingly used for rapid, accurate diagnosis to improve the Outcome of patient. New and existing DNA amplification platforms have high sensitivity and specificity for direct detection and identification of fungi in clinical specimens. Novel technologies (e.g., isothermal and PNA FISH methods), platforms enabling high-throughput analyses (e.g., DNA microarrays and Luminex xMAP) and/or commercial PCR assays are some of advances in diagnosis of Aspergillosis (Spiess B, Seifarth W, Hummel M, Frank O, Fabarius A, Zheng C, Mörz H, Hehlmann R, Buchheidt D. DNA microarray-based detection and identification of fungal pathogens in clinical samples from neutropenic patients. J Clin Microbiol. 2007 November;45(11):3743-53. Epub 2007 Aug. 22).
Unique internal transcribed sequence 2 (ITS2) coding regions have been used to develop nucleic acid probes for different species of Aspergillus (A. flavus, A. fumigatus, A. niger, A. terreus, and A. nidulans), as disclosed in U.S. Pat. No. 6,372,430 (U.S. Pat. No. 6,372,430—Nucleic acids for detecting Aspergillus species and other filamentous fungi). Real time PCR methodologies using ITS region in Aspergilli has also been described for specific detection from clinical samples and Agri products (Schabereiter-Gurtner C, Selitsch B, Rotter M L, Hirschl A M, Willinger B Development of novel real-time PCR assays for detection and differentiation of eleven medically important Aspergillus and Candida species in clinical specimens. J Clin Microbiol. 2007 March;45(3):906-14, Ramirez M, Castro C, Palomares J C, Torres M J, Aller A I, Ruiz M, Aznar J, Martín-Mazuelos E. Molecular detection and identification of Aspergillus spp. from clinical samples using real-time PCR.Mycoses. 2009 March;52(2):129-34, Faber J, Moritz N, Henninger N, Zepp F, Knuf M. Rapid detection of common pathogenic Aspergillus species by a novel real-time PCR approach.Mycoses. 2009 May;52(3):228-33, Bolehovska R, Pliskova L, Buchta V, Cerman J, Hamal P. Detection of Aspergillus spp. in biological samples by real-time PCR. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2006 November;150(2):245-8, Mulè G, Susca A, Logrieco A, Stea G, Visconti A.Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes. Int J Food Microbiol. 2006 Sep. 1;111 Suppl 1:S28-34). Recently a high throughput assay based on Luminex xMAP hybridization technology has been described for clinically relevant fungal pathogens including Aspergillus species (Etienne K A, Kano R, Balajee S A. Development and validation of a microsphere-based Luminex assay for rapid identification of clinically relevant aspergilli. J Clin Microbiol. 2009 April;47(4):1096-100). Monochrome LightCycler real-time PCR based on fluorescence probe have also been described for specific quantification of Aspergillus (Bu R, Sathiapalan R K, Ibrahim M M, Al-Mohsen I, Almodavar E, Gutierrez M I, Bhatia K. Monochrome LightCycler PCR assay for detection and quantification of five common species of Candida and Aspergillus. J Med Microbiol. 2005 March;54(Pt 3):243-248, Imhof A, Schaer C, Schoedon G, Schaer D J, Walter R B, Schaffner A, Schneemann M. Rapid detection of pathogenic fungi from clinical specimens using LightCycler real-time fluorescence PCR.Eur J Clin Microbiol Infect Dis. 2003 Sep;22(9):558-60). Some of the methods such as DNA microarrays are also reported for detection of Aspergillus species from clinical samples (Spiess B, Seifarth W, Hummel M, Frank O, Fabarius A, Zheng C, Mörz H, Hehlmann R, Buchheidt D. DNA microarray-based detection and identification of fungal pathogens in clinical samples from neutropenic patients. J Clin Microbiol. 2007 November;45(11):3743-53) but these are expensive to perform and require sophisticated analytical tools to interpret the results. However all these tests are based on internal transcribed sequence and spacer regions between 28S rRNA and 18srRNA sequences. Genes of important enzymes in the mycotoxin biosynthetic pathway can be good targets for diagnostic tests as it will not only assure the presence of fungus but can also tell us about the mycotoxin production for the same gene which will add to specificity and sensitivity of the test (Baird R, Abbas H K, Windham G, Williams P, Baird S, Ma P, Kelley R, Hawkins L, Scruggs M. Identification of Select Fumonisin Forming Fusarium Species Using PCR Applications of the Polyketide Synthase Gene and its Relationship to Fumonisin Production in vitro. Int J Mol Sci. 2008 April;9(4):554-70, Atoui A, Mathieu F, Lebrihi A. Targeting a polyketide synthase gene for Aspergillus carbonarius quantification and ochratoxin A assessment in grapes using real-time PCR. Int J Food Microbiol. 2007 Apr. 20;115(3):313-8).
In the current invention a diagnostic assay for detection and identification of important Aspergillus species based on the gene of a key enzyme in polyketide biochemical pathway is developed, which will also add to specificity and sensitivity of the test. Potential mycotoxin production can be detected by PCR which may permit the establishment of critical control points and is a significant advantage. Aspergillus species are known to produce a wide range of secondary metabolites, under certain environmental conditions. Some of the important polyketides produced by Aspergillus species include Melanin pigments from A. fumigatus and carcinogenic mycotoxins Aflatoxins from A. flavus. Melanin is considered a virulent factor of Aspergillus fumigatus. Aspergillus flavus is also known to be an opportunistic pathogen of agricultural crops such as maize, cotton, groundnuts, rice, chillies and contaminate them with Aflatoxins and Sterigmatocystin. FAO approved permeable limits of Aflatoxin in agri products are 4-20 ppb in different countries (Jelinek C F, Pohland A E, Wood G E. Worldwide occurrence of mycotoxins in foods and feeds—an update. J Assoc Off Anal Chem. 1989 March-April;72(2):223-30). A. niger is also reported to produce ochratoxins, which contaminates nuts and coffee beans. Ochratoxin A, a polyketide product is teratogenic in rat, hamster and chick embryo and is an inhibitor of hepatic mitochondrial transport systems. It has also been reported to cause damage to the liver, gut, lymphoid tissue and renal tubular damage (Chulze S N, Magnoli C E, Dalcero A M. Occurrence of ochratoxin A in wine and ochratoxigenic mycoflora in grapes and dried vine fruits in South America.Int J Food Microbiol. 2006 Sep. 1; 111 Suppl 1:S5-9).
Diversity in end product produced by polyketide biosynthetic pathway by each Aspergillus species suggests the possible diversity in the structure and function of important enzymes such as Polyketide Synthase. Polykide synthase is key enzyme in the biochemical pathway responsible for production of polyketides and they are highly diverse in Aspergillus species. Polyketide Synthases of Aspergillus species are multidomain and multifuncational proteins of approximately 3000 amino acids with 7 to 9 domains encoded by a single gene (Schümann J, Hertweck C. Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes. J Biotechnol. 2006 Aug. 5; 124(4):690-703, Bhetariya P., Madan T, Varma A., Basir S, Sarma P U. Allergens/Antigens, Toxins and Polyketides of Important Aspergillus Species. Indian Journal of Clinical Biochemistry, June 2011. Vol. 26(2)104-119). The domains of the enzyme facilitate different steps in the synthesis of various intermediates of polyketide products. There are few domains essential for the minimal functional role of PKS while other domains are responsible for post polyketide modifications. Bioinformatics analysis of these Polyketide synthase domain sequences revealed conserved motifs and some non conserved sequences in the PKS domains. Based on the sequence analysis of these Polyketide synthases, sequences were identified and change in the sequences was identified. Information has been used to develop the diagnostic test for Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger in Agriculture samples for detection and identification of Aspergillus infections. Nucleotide sequences are selected and modified and probes have been developed for detection of Aspergillus species relevance to human and agriculture.